Reverse engineering a protein: the mechanochemistry of ATP synthase

ATP synthase comprises two rotary motors in one. The F(1) motor can generate a mechanical torque using the hydrolysis energy of ATP. The F(o) motor generates a rotary torque in the opposite direction, but it employs a transmembrane proton motive force. Each motor can be reversed: The F(o) motor can drive the F(1) motor in reverse to synthesize ATP, and the F(1) motor can drive the F(o) motor in reverse to pump protons. Thus ATP synthase exhibits two of the major energy transduction pathways employed by the cell to convert chemical energy into mechanical force. Here we show how a physical analysis of the F(1) and F(o) motors can provide a unified view of the mechanochemical principles underlying these energy transducers.     

Mechanisms of type III protein export for bacterial flagellar assembly

Flagellar type III protein export is highly organized and well controlled in a timely manner by dynamic, specific and cooperative interactions among components of the export apparatus, allowing the huge and complex macromolecular assembly to be built efficiently. The bacterial flagellum, which is required for motility, consists of a rotary motor, a universal joint and a helical propeller. Most of the flagellar components are translocated to the distal, growing end of the flagellum for assembly through the central channel of the flagellum itself by the flagellar type III protein export apparatus, which is postulated to be located on the cytoplasmic side of the flagellar basal body. The export specificity switching machinery, which consists of at least two proteins that function as a molecular ruler and an export switch, respectively, monitors the state of hook-basal body assembly in the cell exterior and switches export specificity, thereby coupling sequential flagellar gene expression with the flagellar assembly process. The export ATPase complex composed of an ATPase and its regulator acts as a pilot to deliver its export substrate to the export gate and helps initial entry of the substrate N-terminal chain into a narrow pore of the export gate. The energy of ATP hydrolysis appears to be used to disassemble and release the ATPase complex from the protein about to be exported, and the rest of the successive unfolding/translocation process of the long polypeptide chain is driven solely by proton motive force (PMF), perhaps through biased one-dimensional Brownian diffusion. Interestingly, the subunits of the ATPase complex have significant sequence similarities to subunits of F(0)F(1)-ATP synthase, a rotary motor that drives the chemical reaction of ATP synthesis using PMF, and the entire crystal structure of the export ATPase is extremely similar to the alpha/beta subunits of F(0)F(1)-ATP synthase, suggesting that the flagellar export apparatus and F(0)F(1)-ATP synthase share the mechanism for their two distinct functions.     

Mechanism of the F(1)F(0)-type ATP synthase, a biological rotary motor

The F(1)F(0)-type ATP synthase is a key enzyme in cellular energy interconversion. During ATP synthesis, this large protein complex uses a proton gradient and the associated membrane potential to synthesize ATP. It can also reverse and hydrolyze ATP to generate a proton gradient. The structure of this enzyme in different functional forms is now being rapidly elucidated. The emerging consensus is that the enzyme is constructed as two rotary motors, one in the F(1) part that links catalytic site events with movements of an internal rotor, and the other in the F(0) part, linking proton translocation to movements of this F(0) rotor. Although both motors can work separately, they must be connected together to interconvert energy. Evidence for the function of the rotary motor, from structural, genetic and biophysical studies, is reviewed here, and some uncertainties and remaining mysteries of the enzyme mechanism are also discussed.     

Theories of rotary motors

The bacterial flagellar motor and the ATP-hydrolysing F1 portion of the F1Fo-ATPase are known to be rotary motors, and it seems highly probable that the H+-translocating Fo portion rotates too. The energy source in the case of Fo and the flagellar motor is the flow of ions, either H+ (protons) or Na+, down an electrochemical gradient across a membrane. The fact that ions flow in a particular direction through a well-defined structure in these motors invites the possibility of a type of mechanism based on geometric constraints between the rotor position and the paths of ions flowing through the motor. The two best-studied examples of such a mechanism are the ''turnstile'' model of Khan and Berg and the ''proton turbine'' model of Läuger or Berry. Models such as these are typically represented by a small number of kinetic states and certain allowed transitions between them. This allows the calculation of predictions of motor behaviour and establishes a dialogue between models and experimental results. In the near future structural data and observations of single-molecule events should help to determine the nature of the mechanism of rotary motors, while motor models must be developed that can adequately explain the measured relationships between torque and speed in the flagellar motor.     

分子发动机研究进展

分子发动机是利用化学能/化学势进行机械作功的生物大分子,包括线性分子发动机与旋转式分子发动机两大类.它们参与了胞质运输、DNA复制、基因转录、ATP合成／水解等一系列重要生命活动过程.目前对于各种分子发动机的结构及作用机制的研究取得了一些重要进展.     

Energy complexes are apparently associated with the switch-motor complex of bacterial flagella

Recently, the switch-motor complex of bacterial flagella was found to be associated with a number of non-flagellar proteins, which, in spite of not being known as belonging to the chemotaxis system, affect the function of the flagella. The observation that one of these proteins, fumarate reductase, is essentially involved in electron transport under anaerobic conditions raised the question of whether other energy-linked enzymes are associated with the switch-motor complex as well. Here, we identified two additional such enzymes in Escherichia coli. Employing fluorescence resonance energy transfer in vivo and pull-down assays invitro, we provided evidence for the interaction of F(0)F(1) ATP synthase via its β subunit with the flagellar switch protein FliG and for the interaction of NADH-ubiquinone oxidoreductase with FliG, FliM, and possibly FliN. Furthermore, we measured higher rates of ATP synthesis, ATP hydrolysis, and electron transport from NADH to oxygen in membrane areas adjacent to the flagellar motor than in other membrane areas. All these observations suggest the association of energy complexes with the flagellar switch-motor complex. Finding that deletion of the β subunit in vivo affected the direction of flagellar rotation and switching frequency further implied that the interaction of F(0)F(1) ATP synthase with FliG is important for the function of the switch of bacterial flagella.     

A programmable optical angle clamp for rotary molecular motors

Optical tweezers are widely used for experimental investigation of linear molecular motors. The rates and force dependence of steps in the mechanochemical cycle of linear motors have been probed giving detailed insight into motor mechanisms. With similar goals in mind for rotary molecular motors we present here an optical trapping system designed as an angle clamp to study the bacterial flagellar motor and F(1)-ATPase. The trap position was controlled by a digital signal processing board and a host computer via acousto-optic deflectors, the motor position via a three-dimensional piezoelectric stage and the motor angle using a pair of polystyrene beads as a handle for the optical trap. Bead-pair angles were detected using back focal plane interferometry with a resolution of up to 1 degrees , and controlled using a feedback algorithm with a precision of up to 2 degrees and a bandwidth of up to 1.6 kHz. Details of the optical trap, algorithm, and alignment procedures are given. Preliminary data showing angular control of F(1)-ATPase and angular and speed control of the bacterial flagellar motor are presented.     

A structure-based model for the synthesis and hydrolysis of ATP by F1-ATPase

Many essential functions of living cells are performed by nanoscale protein motors. The best characterized of these is F(o)F1-ATP synthase, the smallest rotary motor. This rotary motor catalyzes the synthesis of ATP with high efficiency under conditions where the reactants (ADP, H2PO4(-)) and the product (ATP) are present in the cell at similar concentrations. We present a detailed structure-based kinetic model for the mechanism of action of F1-ATPase and demonstrate the role of different protein conformations for substrate binding during ATP synthesis and ATP hydrolysis. The model shows that the pathway for ATP hydrolysis is not simply the pathway for ATP synthesis in reverse. The findings of the model also explain why the cellular concentration of ATP does not inhibit ATP synthesis.     

ATP synthesis: the world's smallest wind-up toy

ATP synthase contains two rotary motors coupled back-to-back: the protonmotive force-driven motor F0 pushes the ATP-driven motor F1 in reverse, causing it to synthesize ATP. Half of this process has now been reproduced in vitro, using tiny magnets instead of F0 to drive the reverse rotation of a single F1 molecule.     

Elastic deformations of the rotary double motor of single F(o)F(1)-ATP synthases detected in real time by Förster resonance energy transfer

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, F?rster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to a 3-stepped F(1) motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F(O)F(1)-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

ATP synthase: two motors, two fuels

FoF1 ATPase is the universal protein responsible for ATP synthesis. The enzyme comprises two reversible rotary motors: Fo is either an ion 'turbine' or an ion pump, and F1 is either a hydrolysis motor or an ATP synthesizer. Recent biophysical and biochemical studies have helped to elucidate the operating principles for both motors.     

Rotary DNA motors.

Many molecular motors move unidirectionally along a DNA strand powered by nucleotide hydrolysis. These motors are multimeric ATPases with more than one hydrolysis site. We present here a model for how these motors generate the requisite force to process along their DNA track. This novel mechanism for force generation is based on a fluctuating electrostatic field driven by nucleotide hydrolysis. We apply the principle to explain the motion of certain DNA helicases and the portal protein, the motor that bacteriophages use to pump the genome into their capsids. The motor can reverse its direction without reversing the polarity of its electrostatic field, that is, without major structural modifications of the protein. We also show that the motor can be driven by an ion gradient; thus the mechanism may apply as well to the bacterial flagellar motor and to ATP synthase.     

Analysis of bacterial flagellar rotation

Bacterial flagella have rotary motors at their base; embedded in the cytoplasmic membrane and powered by transmembrane ion gradients instead of ATP. Assays have been developed to measure the torque output of individual motors over a wide regime of load, to correlate the energizing proton flux with rotation speed and relate through genetic analysis motor structure to function. These assays promise substantial advances in understanding mechanochemical coupling in these motors. Here, I summarize the present status of our understanding of energy transduction in bacterial flagella and compare this with the case for muscle.     

Distinct in situ structures of the Borrelia flagellar motor

Bacteria can be propelled in liquids by flagellar filaments that are attached to and moved by flagellar motors. These motors are rotary nanomachines that use the electrochemical potential from ion gradients. The motor can spin in both directions with specific proteins regulating the direction in response to chemotactic stimuli. Here we investigated the structure of flagellar motors of Borrelia spirochetes, the causative agents of Lyme disease in humans. We revealed the structure of the motor complex at 4.6-nm resolution by sub-volume averaging of cryo-electron tomograms and subsequently imposing rotational symmetry. This allowed direct visualisation of individual motor components, the connection between the stator and the peptidoglycan as well as filamentous linkers between the stator and the rod. Two different motor assemblies seem to co-exist at a single bacterial pole. While most motors were completely assembled, a smaller fraction appeared to lack part of the C-ring, which plays a role in protein export and switching the directionality of rotation. Our data suggest a novel mechanism that bacteria may use to control the direction of movement.     

The rotary binding change mechanism of ATP synthases

The F(0)F(1) ATP synthase functions as a rotary motor where subunit rotation driven by a current of protons flowing through F(0) drives the binding changes in F(1) that are required for net ATP synthesis. Recent work that has led to the identification of components of the rotor and stator is reviewed. In addition, a model is proposed to describe the transmission of energy from four proton transport steps to the synthesis of one ATP. Finally, some of the requirements for efficient energy coupling by a rotary binding change mechanism are considered.     

Inter-subunit rotation and elastic power transmission in F0F1-ATPase

ATP synthase (F-ATPase) produces ATP at the expense of ion-motive force or vice versa. It is composed from two motor/generators, the ATPase (F1) and the ion translocator (F0), which both are rotary steppers. They are mechanically coupled by 360 degrees rotary motion of subunits against each other. The rotor, subunits gamma(epsilon)C10-14, moves against the stator, (alphabeta)3delta(ab2). The enzyme copes with symmetry mismatch (C3 versus C10-14) between its two motors, and it operates robustly in chimeric constructs or with drastically modified subunits. We scrutinized whether an elastic power transmission accounts for these properties. We used the curvature of fluorescent actin filaments, attached to the rotating c ring, as a spring balance (flexural rigidity of 8.10(-26) N x m2) to gauge the angular profile of the output torque at F0 during ATP hydrolysis by F1. The large average output torque (56 pN nm) proved the absence of any slip. Angular variations of the torque were small, so that the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the three-fold stepping and high activation barrier (>40 kJ/mol) of the driving motor (F1) itself, the rather constant output torque seen by F0 implied a soft elastic power transmission between F1 and F0. It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate under load of the two counteracting and stepping motors/generators.     

Electrostatic interactions in catalytic centers of F1-ATPase

The first part of this paper is a brief review of works concerned with the mechanisms of functioning of F0F1-ATP synthases. F0F1-ATP syntheses operate as rotating molecular machines that provide the synthesis of ATP from ADP and inorganic phosphate (Pi) in mitochondria, chloroplasts, and bacteria at the expense of the energy of electrochemical gradient of hydrogen ions generated across energy-transducing mitochondrial, chloroplast or, bacterial membranes. A distinguishing feature of these enzymes is that they operate as rotary molecular motors. In the second part of the work, we calculated the contribution of electrostatic interactions between charged groups of a substrate (MgATP), reaction products (MgADP and Pi), and charged amino acid residues of the F1-ATPase molecule to energy changes associated with the binding of ATP and its chemical transformations in the catalytic centers located at the interface of the alpha- and beta-subunits of the enzyme (oligomer complex alpha 3 beta 3 gamma of bovine mitochondrial ATPase). The catalytic cycle of ATP hydrolysis considered in the work includes conformational changes of alpha- and beta-subunits caused by unidirectional rotations of the central gamma-subunit. The results of our calculations are consistent with the idea that the energetically favorable process of ATP binding to the "open" catalytic center of F1-ATPase initiates the rotation of the gamma-subunit followed by ATP hydrolysis in another ("closed") catalytic center of the enzyme.     

Quantification of flagellar motor stator dynamics through in vivo proton‐motive force control

The bacterial flagellar motor, one of the few rotary motors in nature, produces torque to drive the flagellar filament by ion translocation through membrane‐bound stator complexes. We used the light‐driven proton pump proteorhodopsin (pR) to control the proton‐motive force (PMF) in vivo by illumination. pR excitation was shown to be sufficient to replace native PMF generation, and when excited in cells with intact native PMF generation systems increased motor speed beyond the physiological norm. We characterized the effects of rapid in vivo PMF changes on the flagellar motor. Transient PMF disruption events from loss of illumination caused motors to stop, with rapid recovery of their previous rotation rate after return of illumination. However, extended periods of PMF loss led to stepwise increases in rotation rate upon PMF return as stators returned to the motor. The rate constant for stator binding to a putative single binding site on the motor was calculated to be 0.06 s^?1. Using GFP‐tagged MotB stator proteins, we found that transient PMF disruption leads to reversible stator diffusion away from the flagellar motor, showing that PMF presence is necessary for continued motor integrity, and calculated a stator dissociation rate of 0.038 s^?1.     

Rotation and structure of FoF1-ATP synthase

F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background.